1. Field of the Invention
The present invention relates to a microscope device, which is capable of producing images of a sample in different spectral ranges (“colors”) or polarisations on a single detector.
2. Description of Related Art
In 1991 a method for separating a microscope image into two images of different color placed next to each other on a single camera chip was proposed (K. Kinosita et al., “Dual-View Microscopy with a Single Camera: Real-Time Imaging of Molecular Orientations and Calcium”, J. Cell. Biol., 1991, 115(1), pages 67-73); see also U.S. Pat. No. 5,337,081). The commercial version of this concept was termed “w-View” because it not only provides a “double view” of the sample, but also because of the w-shape of the beam path it employs. (http://jp.hamamatsu.com/resources/products/sys/pdf/eng/e_aqfret.pdf). The design is used in all fields of microscopy where dual-emission images need to be recorded. Given that it allows recording two color images simultaneously rather than sequentially, the device is particularly suited for time-lapse studies, where switching of an emission filter would reduce time resolution.
A schematic of the W-view design as used in the prior art is shown in FIG. 1. A collimated image beam 10, originating from an intermediate image (which is not shown in FIG. 1) impinges onto a first dichroic beam splitter 12 and is separated into a first beam 14 and a second beam 16 of different color, i.e. the first beam 14 essentially consists of light of a first spectral range and the second beam 16 essentially consists of light of a second spectral range. The first beam 14, which is reflected by the first beam splitter 12, passes to a mirror 18 from where it is directed onto a second dichroic beam splitter 20, which has the same spectral characteristics as the first beam splitter 12. The second beam 16, which is transmitted by the first beam splitter 12, is reflected by a second mirror 22 and directed onto the second beam splitter 20, where the first beam 14 and the second beam 16 are “reunited” (or “combined”) in their general direction. However, by adjusting the mirrors 18 and 22 in an appropriate fashion, the two beams 21, corresponding to the first beam 14, and 27, corresponding to the second beam 16, exhibit a slight angular offset relative to each other, i.e. they diverge relative to each other to a certain degree, so as to yield the desired spatial separation on the detector chip (not shown in FIG. 1).
According to this prior art concept, color separation takes place in an infinity space of the optical beam path, which could be the space between the objective lens and the tube lens. However, in order to avoid image overlap from adjacent areas, it is advantageous to create an intermediate image and to confine it within boundaries defined by a suitable field-stop. Such field-stop has to reduce the field of view seen by the camera of the detector to one half of its original size, in order to accommodate the two semi-images projected side by side. Having an intermediate image requires a second set of optics (relay-lenses), which create another infinity space where beam separation takes place. One major advantage of this design is that using a beam splitter not only for separating beams, but also for reuniting them, allows maintaining telecentric optics throughout, thus avoiding vignetting and asymmetrical light-cones which are different for different areas on the detector chip. Moreover, the fact that both beam paths are transmitted by the same optics warrants that both color channels experience identical magnification and need no resealing before being compared. This is particularly important in co-localization studies.
However, since no optical system is perfect, there are always aberrations and distortions, and their extent depends on the position of a given point within the field. Usually, aberrations are less pronounced in the center and increase towards the edges of the field. However, due to the spherical symmetry of the imaging optics, aberrations and distortions are generally symmetrical with respect to the central axis of the optics.
According to U.S. Pat. No. 5,982,479 a single dichroic beam splitter may be used for separating a collimated image beam originating from an intermediate image into two different color channels, which are imaged by a common lens onto a common detector chip in order to obtain spatially separated semi-images on the detector. Each of the color channels is reflected twice prior to being projected onto the detector, whereas according to the prior art system shown in FIG. 1, both color channels are reflected 3 times or, in the original Kinosita paper, one color channel is not reflected at all whereas the other one is reflected 4 times A similar system is known from JP 2004361391 A, wherein splitting of the two color channels and double-reflection in each channel occurs in the space between the projection lens and the detector. All prior art has in common that the number of reflections for the two beam-paths are such that both color channels have the same handedness on the chip.
It is an object of the invention to provide for a microscope device having dual emission capability, wherein detrimental effects of image-aberrations and -distortions are reduced.